Cell Meter™ Intracellular NADH/NADPH Fluorescence Imaging Kit *Red Fluorescence*

Protocol summary

1. Prepare cells in growth medium
2. Incubate cells with test compounds and JZL1707 NAD(P)H Sensor working solution at 37^oC 30 - 60 minutes 
3. Wash and keep cells in Assay Buffer
4. Monitor fluorescence intensity (bottom read mode) at Ex/Em = 540/590 nm (Cutoff = 570 nm) or fluorescence microscope with TRITC filter

Important
Thaw all the kit components at room temperature before starting the experiment.

Add 10 µL of JZL1707 NAD(P)H Sensor stock solution (Component A) into 2.5 mL of Assay Buffer (Component B), and mix well to make JZL1707 NAD(P)H Sensor working solution. This JZL1707 NAD(P)H Sensor working solution is stable within 1 hour at room temperature. Note: 40 µL of JZL1707 NAD(P)H Sensor stock solution is enough for one plate. Protect from light.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

1. To stimulate NADP/NADPH, treat cells with 10 µL of 10X test compounds (96-well plate) or 5 µL of 5X test compounds (384-well plate) in serum free medium or your desired buffer (such as PBS or HHBS). For control wells (untreated cells), add the corresponding amount of medium or compound buffer. Note: JZL1707 NAD(P)H Sensor is serum sensitive, therefore it’s recommended to keep cells in serum-free medium or the buffer of your choice. Alternatively, cells can be prepared and treated in regular full medium. Change to serumfree medium or buffer of your choice when incubation with JZL1707 NAD(P)H Sensor.                                                                                                                                                                                             
2. Add 100 µL/well (96-well plate) or 25µL/well (384-well plate) of JZL1707 NAD(P)H Sensor working solution in the cell plate. Co-incubate cells with test compound and JZL1707 NAD(P)H Sensor working solution at 37^oC for 30-60 minutes, protected from light. Note: For a NADH/NADPH positive control treatment: HeLa cells were incubated with 100 µM NADH or NADPH for 30 minutes in serum-free medium, and co-incubated with JZL1707 NAD(P)H sensor working solution at 37^oC for another 30 minutes. See Figure 1 for details.
   
   
3. Wash cells with your desired buffer once. Remove solution in each well and add Assay Buffer (Component B) 100 µL/well for a 96-well plate or 25 µL/well for a 384-well plate.
   
   
4. Monitor the fluorescence increase using microplate reader at Ex/Em = 540/590 nm (Cutoff = 570 nm) with bottom read mode, OR take images using fluorescence microscope with the filter set of Cy3® filter or TRITC.